Expression of wound healing and stress-related proteins in keratoconus corneas

PURPOSE: Keratoconus is characterized by thinning and scarring of the central portion of the cornea. This study was performed on keratoconus corneas to examine the expression of proteins related to wound healing including vimentin, an intermediate filament protein, and tenascin, and extracellular matrix protein. The expression of stress-related cytokines, heat shock proteins and ubiquitin was also investigated. METHODS: Corneal buttons were collected from patients with keratoconus, normal subjects and patients with other corneal diseases such as pseudophakic bullous keratopathy. Immunofluorescence staining was performed on frozen sections for vimentin and tenascin, and immunoperoxidase staining was carried out on paraffin sections for cytokines, heat shock proteins and ubiquitin. RESULTS: To varying degrees, all proteins examined, except tenascin and heat shock protein 90, were found to be expressed in normal human corneas. The expression of vimentin, tenascin, transforming growth factor-beta, interleukin-1, heat shock protein 27, and ubiquitin was enhanced in keratoconus corneas. A similar enhancement however was also observed in other diseased corneas. CONCLUSIONS: Altered expression of several wound healing or stress-related proteins was noted in keratoconus corneas. The alterations appear to be nonspecific injury or wound responses in association with corneal diseases.     

Basal membrane heparan sulphate proteoglycan expression during wound healing in human skin

Heparan sulphate proteoglycans (HSPGs) are integral components of the basement membrane (BM) in various tissues. HSPGs are important in the assembly and structure of the BM, and their putative functions include regulation of basement membrane permeability, binding of growth factors, and a role in cellular adhesion. In this study the expression of HSPG was examined during wound healing in human skin, using monoclonal antibodies (MAbs) that recognize the HSPG core protein and two different heparan sulphate (HS) epitopes, and the dynamics of HSPG expression were investigated in relation to epidermal cellular proliferation and permeability of the BM. Healing of excisional wounds in healthy volunteers was studied from day 0 up to 1 year. Intact human skin showed strong continuous staining of the dermo-epidermal BM and the vascular BM with all MAbs. Up to day 4 after wounding, staining for HSPG was absent under the ingrowing epidermis, with any of the MAbs, indicating that no complete BM was present. From day 7 onwards, the BM of the neo-epidermis showed positive staining for the HSPG core protein and a low sulphated HS epitope, and after day 14, the staining intensity was similar to normal skin. The staining patterns of these HSPG epitopes were similar to that of laminin. The staining pattern with a MAb against an epitope in the highly sulphated part of HS was found to be distinct from the other BM markers studied. This epitope was absent under the neo-epidermis up to 2 months after wounding. One year after wounding, the epitope was found to be present again. We observed that only in the time period between 2 months and 1 year had the epidermis normalized with respect to the number of cycling cells and the absence of high molecular weight plasma proteins. These findings suggest a correlation between normalization of epidermal proliferation, BM permeability, and regeneration of BM HS. It is proposed that complete BM maturation following skin wounding is a slow process and may account for the epidermal abnormalities that persist for a considerable period of time after wound healing.     

Expression of Wnt genes in early wound healing

The effects of ambient temperature (22 degrees C/10 degrees C) and season (summer/winter) on anuran skin allograft and xenograft rejection was tested in frogs (Rana temporaria and R. esculenta) and toads (Bufo bufo, Bombina variegata, and Bombina bombina). Mean graft survival times were significantly prolonged at the low temperature in a species-specific manner, the edible frog (R. esculenta) being the most sensitive and the common toad (Bufo bufo) relatively resistant. Allografts were more temperature-dependent than xenografts; in the latter case, temperature sensitivity was specific to each donor-host combination. Rejection of second-set grafts in R. esculenta was accelerated both in warmth and in cold, but second-set grafts were less temperature-sensitive than sensitizing ones. Both in summer and in winter, R. esculenta rejected allografts promptly at 22 degrees C but slowly at 10 degrees C. In both seasons, Bombina variegata kept at 22 degrees C rejected allografts in a chronic manner. This indicates that amphibian transplantation immunity depends on the donor-host genetic disparity and ambient temperature but is independent of season.     

Is heat shock protein re-induction during tolerance related to the stressor-specific induction of heat shock proteins?

The existence of stressor-specific induction programs of heat shock proteins (hsps) leads us to analyze the possible occurrence of a stressor-specific tolerance induced by either heat shock, arsenite, or cadmium. As a measure of this tolerance re-induction of hsps was studied. In this paper, we tested whether the refractory state is either valid for each specific hsp (implying independent regulation of every member of the heat shock protein family) or extends from small subsets of the hsp-family to even larger groups of proteins (indicating a more common denominator in their regulation). (re-)induction of hsps does not seem to be regulated at the level of each individual hsp since differences in induced synthesis of hsps between two stressor conditions are not supplemented systematically upon the sequential application of the two stressors. The most notable example in this respect is hsp60. A pretreatment with cadmium, which hardly induces synthesis of this hsp, does induce a tolerance to (re)-induction by heat shock, which normally induces hsp60. This suggests the existence of a more common denominator regulating the coordinate expression of at least some hsps. From our data we conclude that the degree, but not the pattern, of hsp re-induction is influenced by the type of stressor used in the pretreatment. The pattern of hsps induced by a secondary applied stressor still shows most of its stressor-specificity and seems to be independent of any pretreatment. The possible implications of stressor-specificity are discussed.     

Monoclonal antibodies to mammalian heat shock proteins impair mouse embryo development in vitro

Two-cell mouse embryos (B6D2F1) were cultured in the presence or absence of 100 microg/ml monoclonal antibodies specific for the mammalian 60 kDa (HSP60), 70 kDa (HSP70) and 90 kDa (HSP90) heat shock proteins. Embryo development was evaluated after 3, 5 and 7 days in culture by determining the number of blastocysts, hatched blastocysts and outgrown trophoblasts at the successive time points. At day 3, only 29% (22/75) of the embryos cultured with anti-HSP60 antibody developed to the blastocyst stage (P < 0.0001) as compared to 67% (31/46) of the embryos cultured with anti-HSP70, 72% (43/60) cultured with anti-HSP90, and 79% (49/62) in medium plus mouse IgG1. By day 5, hatched embryos were present in 28% (13/ 46) of the cultures containing anti-HSP70 (P < 0.0001), as opposed to 57% (34/60) containing anti-HSP90 and 73% (45/62) containing IgG1. At day 7, outgrown trophoblasts were observed in 9% (4/46) of cultures containing anti-HSP70 (P < 0.0001), 45% (27/60) containing anti-HSP90 (P < 0.01) and 66% (41/62) cultured in medium plus IgG1. Antibodies to different heat shock proteins exerted a detrimental effect on mouse embryo development at unique development stages. Immune sensitization to heat shock proteins may be a cause of reproductive failure.     

Expression of Msx-2 during development, regeneration, and wound healing in axolotl limbs

BACKGROUND: High exposure to house dust mite allergen during the first year of life has been found to increase the risk of subsequent asthma and mite sensitization. Environmental factors, home construction and cleaning methods used are associated with levels of dust mites in the home. OBJECTIVE: To investigate determinants of levels of Der p 1 and Der f 1 mite allergens in homes of infants in southern Tasmania. METHODS: Dust samples were collected from 72 homes of infants participating in the Tasmanian Infant Health Survey (TIHS). The Der p 1 and Der f 1 allergen concentrations in these samples were measured. The TIHS interviewers obtained information from the mothers of the infants via a questionnaire, observed specified aspects of the home environment, and took readings of bedroom temperature and humidity. The effect of each item on allergen concentration in dust from bedroom floors was examined in a variety of ways. Those items which in this study appeared to be significantly related to allergen concentrations plus items which in other studies have been found to be related to allergen concentrations were then investigated further in multivariate models. RESULTS: Der p 1 allergen concentration (microg/g) and density (microg/m2) in dust from bedroom floors were found to be related to several home environment factors. In the univariate analyses, indoor humidity, 24 h maximum temperature, number of residents and a combination of floor covering and cleaning methods appeared to have a significant effect on allergen levels. These factors remained important in the multivariate model except that indicators for mould in the bathroom and drying washing on an outside line replaced indoor humidity. CONCLUSION: Features related to home dampness, the number of residents and floor covering and cleaning were major determinants of Der p 1 levels in the bedrooms studied.     

Effect of Tissucol on connective tissue matrix during wound healing: an immunohistochemical study in rat skin

OBJECTIVE: Our goal was to identify prenatally available parameters that correlate with neonatal outcome and could be used for predicting such outcome in the extremely low-birth-weight pregnancy. STUDY DESIGN: From 1990 through 1995, obstetric and neonatal data of live-born nonanomalous singleton infants with birth weights between 400 and 1000 gm were reviewed. Only cases in which ultrasonographic biometry, including biparietal diameter, abdominal circumference, and femur length, was performed < or =3 days before delivery were included. Overall survival (defined as alive at discharge) and survival without specific severe neonatal morbidities (namely, retinopathy of prematurity [stage 3 or 4], intraventricular hemorrhage [grade 3 or 4], periventricular leukomalacia, chronic lung disease, and deafness) were ascertained. The best combination of prenatal parameters for the prediction of overall survival and survival without severe morbidity was determined by backward stepwise logistic regression analyses. RESULTS: The most significant prenatal predictors of overall survival were the obstetric estimate of gestational age and the abdominal circumference (chi2 = 11.8036, p = 0.0006 and chi2 = 8.1862, p < 0.005, respectively). Survival without severe morbidity was also predicted by the same combination of parameters (chi2 = 21.9079, p = 0.0001 and chi2 = 6.538, p = 0.01, respectively). The estimated fetal weight was not a significant independent predictor of either category of outcome (chi2 = 0.1249, p = 0.72 and chi2 = 0.0361, p = 0.85, respectively). On the basis of the regression formulas, curves displaying the probabilities of overall survival and survival without severe morbidity with any combination of gestational age and abdominal circumference were developed. CONCLUSION: The combination of gestational age and the abdominal circumference measurements appears to be superior to any combination that included estimated fetal weight data for predicting neonatal outcome in the neonates weighing < or =1000 gm. We developed a mechanism for predicting neonatal outcome in this weight category on the basis of prenatally available parameters. This information could prove useful for both parental counseling and obstetric decision making.     

The utilization of nutrient substances during wound healing

The process of wound healing represents a series of complex physicochemical reactions requiring different nutritional microcomponents at each stage. In patients with extremely grave diseases and injuries the course of wound healing is impaired because of a hypermetabolic reaction to stress, leading to protein catabolism. The hypothalamus responds to cytokine stimulation by changes of thermoregulation (increase of heat production) and increased production of stress hormones (catecholamines, hydrocortisone, and glucagon). In turn, stress hormones trigger the thermogenous (unproductive) metabolic cycle and the lipolysis and proteolysis processes. Hyperproduction of glucose at the expense of skeletal muscle tissue degradation leads to the formation of amino acid substrate for liver glyconeogenesis. Additional nutrients are obligatory for wound healing in such patients. Protein catabolism cannot be arrested by amino, acids alone partly because amino acid transport is impaired; it can be normalized by anabolics, such as growth hormone and insulin-like growth factor 1. Treatment with growth hormone yields a dramatic positive effect in severely burned children. Proteins and vitamins, specifically arginine and vitamins A, B, and C provide the optimal nutritive support during wound treatment.     

Autoantibodies to small heat shock proteins in glaucoma

PURPOSE: To identify the low-molecular-weight retinal proteins that are the targets of serum autoantibodies in patients with glaucoma and to study the ability of these antibodies to induce retinal apoptosis. METHODS: Serum immunoreactivity against retinal proteins was examined in age-matched groups of 60 patients with normal-pressure glaucoma, 36 patients with high-pressure glaucoma, and a control group of 20 healthy subjects, by means of western blot analysis and enzyme-linked immunosorbent assay. The specificity of the immunoreactivity to small heat shock proteins, including alpha-crystallins and hsp27, was tested by immunoprecipitation of these proteins in retinal fractions. The direct effects of antibodies specific to small heat shock proteins were then studied in isolated intact human retina (ex vivo) and cultured rat retinal cells (in vitro) by immunocytochemistry and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) technique in conjunction with fluorescence microscopy and confocal imaging. RESULTS: Serum immunoreactivity against retinal proteins with low molecular weight in patients with glaucoma was to small heat shock proteins, including alpha-crystallins and hsp27. In addition, patients with normal pressure glaucoma had a higher titer of autoantibodies to small heat shock proteins than did age-matched patients with high-pressure glaucoma or control subjects. It was observed that when antibodies against small heat shock proteins were applied directly to retina tissue or cells, they could trigger cell death through an apoptotic mechanism. CONCLUSIONS: These findings suggest that increased titers of circulating antibodies against retinal small heat shock proteins may have pathogenic significance in some patients with glaucomatous optic neuropathy.     

Expression of heat shock protein 27 in chromomycosis

We report on a 58-year-old woman with long-lasting (36 years) chromomycosis on the foot and secondary self-inoculation from foot to hand 4 years ago. Mycological classification was performed after culture on Sabouraud glucose agar. We used haematoxylin and eosin and Giemsa staining and an antibody to heat shock protein (HSP) 27 (Stress Gen, Clone G3.1) on paraffin-embedded and cryostat specimens of chromomycosis. The mycological culture revealed the fungus Fonsecaea pedosoi. Histopathology revealed dermal fibrosis with persistent fungi (Medlar bodies), numerous mast cells and pseudoepitheliomatous hyperplasia. Immunohistochemically, HSP 27 was positively identified in F. pedrosoi. Moreover, in differentiating keratinocytes in the pseudoepitheliomatous lesions of chromomycosis, HSP 27 was increasingly expressed from basal layers to stratum spinosum in the epidermis but not in keratinocytes directly bordering Medlar bodies. In chromomycosis, HSP 27 is expressed, in accordance with its role as a marker of differentiation and proliferation, in keratinocytes and also in F. pedrosoi. It remains unknown if these results might explain the therapeutic efficacy of hyperthermic treatment.     

Role of heat shock proteins in heart transplant rejection

STUDY OBJECTIVE: To study secular trends of placental abruption (PA), the effects of demographic variables and the use of cesarean section (CS) associated with PA. DESIGN: A population based cohort study. SETTING: The Medical Birth Registry of Norway. PATIENTS: 9,592 cases of PA of a total of 1,446,154 births notified, i.e. all births in Norway 1967-1991. MAIN RESULTS: The PA proportion was 6.6 per 1000 births of a gestational age of 16 weeks or more, ranging from 5.3 in 1971 to 9.1 in 1990. Birth order two had the lowest proportion and it increased by maternal age. The PA proportion decreased by gestational age from 86.4 per 1000 below 28 weeks to 3.4 in term pregnancies. The PA proportion per 10,000 pregnancies at risk increased from 1.3 in the 28th week to 14.1 in the 42nd week. A secular trend of a decreasing but still high relative risk of PA in SGA-births at any gestational age increased from 1967 through 1991. The relative risk of PA of Apgar score <7 after five minutes, adjusted for gestational age, was 7.8. CONCLUSIONS: Inspite of an increasing CS rate, an increasing proportion of PA was noted from 1967 through 1991. The proportion was lowest for birth order two and increased by maternal age. To an increasing extent, PA births were centralized. SGA, prematurity and asphyxia were major problems associated with PA. A tendency towards larger infants and a decreasing relative risk of PA in SGA-births might be attributable to improvements in antenatal care.     

Enhanced biosynthesis of extracellular matrix proteins and TGF-beta 1 by polyinosinic-polycytidylic acid during cutaneous wound healing in vivo

OBJECTIVES: We sought to determine the utility of left ventricular expansion velocities in differentiating constrictive pericarditis from restrictive cardiomyopathy. BACKGROUND: Several studies have shown that left ventricular diastolic expansion is influenced by the elastic recoil forces of the myocardium. These forces are affected by intrinsic myocardial disease but should be preserved when diastole is impaired as a result of extrinsic causes. METHODS: Using Doppler tissue imaging, we measured peak early velocity of longitudinal axis expansion (Ea) in 8 patients with constrictive pericarditis, 7 patients with restriction and 15 normal volunteers. Transmitral early (E) and late (A) Doppler flow velocities, left ventricular systolic and diastolic volumes, ejection fraction and mitral annular M-mode displacement were also compared between the groups. RESULTS: The Ea value was significantly higher in normal subjects (14.5 +/- 4.7 cm/s [mean +/- SD]) and in patients with constriction (14.8 +/- 4.8 cm/s) than in those with restriction (5.1 +/- 1.4 cm/s, p < 0.001 constriction vs. restriction). There was weak correlation between Ea and the extent of annular displacement (r = 0.55, p = 0.004) and the E/A ratio (r = 0.44, p = 0.03). There was no correlation between Ea and E (r = 0.33, p = 0.07) or ejection fraction (r = 0.21, p = 0.26). By multivariate analysis, Ea was the best variable for differentiating constriction from restriction. CONCLUSIONS: Our study indicates that longitudinal axis expansion velocities are markedly reduced in patients with restrictive cardiomyopathy. The poor correlation found with transvalvular flow velocities suggests that Ea may be relatively preload independent. The measurement of longitudinal axis expansion velocities provides a clinically useful distinction between constrictive pericarditis and restrictive cardiomyopathy and may prove to be valuable in the study of diastolic function.